Use of a flavonoid extract of ginkgo biloba substantially devoid of terpenes, in the dentibuccal field, and composition containing such extract

ABSTRACT

The invention concerns the use of a flavoniodic extract of ginkgo biloba leaves, and more specifically an extract devoid of terpenes for oral hygiene. The invention also concerns a composition for oral hygiene containing such an extract.

PRIOR APPLICATIONS

This application is a division of U.S. patent application Ser. No.09/284,125 filed Apr. 6, 1999 now U.S. Pat. No. 6,159,450 which is a 371of PCT/FR97/01910 filed Oct. 24, 1997.

The invention relates to the use of a flavonoid extract of ginkgobiloba, and more specifically an extract substantially devoid ofterpenes, in the dentibuccal field. The invention also relates to andentibuccal composition containing such extract.

A flavonoid extract of ginkgo biloba leaves devoid of terpenes accordingto the invention comprises flavonoid glycosides and little or noterpenes. When the extract contains terpenes, the terpene content is 1%maximum, and preferably 0.5% maximum. This extract contains from 28 to35% of flavonoid glycosides, and preferably 28 to 32%. Such extracts arepreferably obtained from leaves from pruned young ginkgo biloba trees.

A subject of the invention is a process for obtaining such an extract, aprocess which comprises several extraction stages of ginkgo bilobaleaves by solvents and characterized in that one of the extractionstages is a deterpenation stage and the solvent used is a compound offormula RC(O)OR′ in which R and R′ represent, independently, a loweralkyl, alone or mixed with a saturated aliphatic hydrocarbon containingat least 5 carbon atoms. The extraction stage can be carried out at anystage in the process. Preferably, the solvent used during thedeterpenation stage contains from 0 to 20% of saturated aliphatichydrocarbon.

Extraction stages other than the deterpenation stage are known in theliterature in particular in Patents EP431535, EP 431536, EP 360556 andEP324197. These Patents are incorporated into the present Application byway of reference.

In the definitions indicated above, the expression lower alkylpreferably represents a linear or branched alkyl radical having from 1to 6 carbon atoms, and in particular an alkyl radical having from 1 to 4carbon atoms such as the methyl, ethyl, propyl, isopropyl, butyl,isobutyl, sec-butyl and tert-butyl radicals. The solvents of formulaRC(O)OR′ in which the radicals R and R′ represent methyl, ethyl orpropyl and in particular ethyl acetate are preferably used. Thesaturated aliphatic hydrocarbon can be chosen from hexane, heptane andoctane. Preferably, heptane is used.

A flavonoid extract of ginkgo biloba leaves as defined above has ananti-elastasic and anti-inflammatory activity on human gums. Anillustration of these properties is to be found below in theexperimental part.

These properties render the ginkgo extract as defined above suitable forpharmaceutical use. A subject of the present Application is therefore,as a medicament, and in particular as a medicament intended for thedentibuccal sphere, a flavonoid extract of ginkgo biloba leaves asdefined above.

A subject of the invention is also a pharmaceutical composition intendedin particular for the treatment of the dentibuccal sphere and containingas active ingredient a flavonoid extract of ginkgo biloba leaves asdefined above, optionally combined with a pharmaceutically acceptablesupport.

A more particular subject of the invention is an anti-elastasicpharmaceutical composition containing, as an anti-elastasic agent, atleast one effective quantity of a ginkgo extract as defined above. Amore particular subject of the invention is also an anti-inflammatorypharmaceutical composition containing, as an anti-inflammatory agent, atleast one effective quantity of a ginkgo extract as defined above.

A pharmaceutical composition according to the invention preferablycontains from 0.05 to 0.6% of ginkgo extract, and preferentially from0.1 to 0.5%. Such a composition can also contain at least one otheragent having similar or complementary activities. Preferably, apharmaceutical composition according to the invention contains ginkgoextract as defined above combined with ceramides. Preferentially, such acomposition contains from 0.1 to 0.5% of ginkgo extract and from 0.05 to0.3% of ceramides.

A pharmaceutical composition according to the invention can be presentedin any appropriate form according to the chosen administration method.It can be presented in liquid form such as, for example, a solution, asuspension or an emulsion. It can also be presented in the form of agel, spray, toothpaste or chewing gum. It is preferably presented in theform of a gel or chewing gum. It can contain, in addition to the ginkgoextract, alone or in combination, and an appropriate support, theingredients or adjuvants commonly used for producing such compositions.

A subject of the invention is also the use of a flavonoid extract ofginkgo as defined above for the preparation of a pharmaceuticalcomposition intended for treatment in the dentibuccal sphere.

A more particular subject of the invention is the use of a flavonoidextract of ginkgo as defined above for the preparation of apharmaceutical composition intended to inhibit elastase. A moreparticular subject of the invention is also the use of a flavonoidextract of ginkgo as defined above for the preparation of pharmaceuticalcomposition intended to inhibit inflammation in the dentibuccal sphere.Such a flavonoid extract can be used alone or in combination withanother agent having similar or complementary properties according tothe sought result, optionally in the presence of an appropriate support.An extract as defined above is preferably used in combination withceramides.

Ceramides are sphingolipids present in all cell structures. They arealso notably present in the plant world and in particular in wheat,rice, soya, millet and spinach. They can also be synthsized. Owing totheir structure, they can advantageously be used as a vector,considerably increasing the bioavailability of the active agent they areconveying. The term ceramide used in the present Application has thestandard meaning known to a person skilled in the art. The term ceramidethus includes all ceramides, of synthetic or natural origin (vegetable,animal or human) optionally substituted, for example, by a sugar such asmono- or polyglucosylceramides.

Owing to a high flavonoid glycoside content, the state of the artsuggests that an extract as defined above has an anti-oxidant andanti-radicular activity. Moreover, given on the one hand itsanti-inflammatory, anti-elastasic properties but also vasoregulatory andcollagen activating properties and, on the other hand, an almost totalabsence of terpenes in its composition, such an extract canadvantageously be used in cosmetics or in dermatology.

A subject of the invention is also the use of a flavonoid extract ofginkgo biloba leaves as defined above for the preparation of avasoregulatory and/or collagen activating medicament. The invention alsorelates to the use of a cosmetic composition containing a flavonoidextract as defined above as a vasoregulator and/or a collagen activator.A subject of the invention is also a cosmetic treatment methodcharacterized in that a flavonoid extract is used as defined above as avasoregulator and/or a collagen activator.

A subject of the invention is also a cosmetic composition intended inparticular for the dentibuccal sphere and containing a flavonoid extractof ginkgo biloba leaves as defined above. This extract can be used onits own or in combination with at least one other agent having a similaror complementary activity, and optionally with an appropriate support.Preferably, the ginkgo extract is used in combination with ceramides. Acosmetic composition according to the invention can contain from 0.05 to0.6% of ginkgo extract as defined above, and from 0.05 to 0.3% ofceramides. Such a composition can be presented in any appropriate formfor this type of use such as creams, emulsions, milks, gels, oils,makeup products, lipsticks and lotions. In addition to ginkgo extract,alone or in combination, and an appropriate support, it can contain theingredients or adjuvants commonly used to produce such compositions.

A subject of the invention is also a flavonoid extract of ginkgo bilobaas defined above for the cosmetic treatment of affections in thedentibuccal sphere and in particular gingivitis.

The following examples are presented in order to illustrate theinvention and should in no event be considered to limit the scope of theinvention.

EXPERIMENTAL PART

Process for obtaining a flavonoid extract

Ginkgo biloba leaves are extracted with from 6 to 12 parts (preferably 8parts) of water containing 60% acetone at 50-60° C. and the solution isconcentrated in order to reduce the percentage of acetone to less than3%. This solution is cooled down and the lipids are eliminated bydecanting. The aqueous solution is extracted with 2 to 5 parts of ethylacetate containing from 0 to 20% heptane. The resultant solution isextracted with a minimum quantity of an acetone/butanol mixture(percentage acetone 0 to 15%) in the presence of ammonium sulphate. Theorganic phase is concentrated; after ethanol has been added, thesolution is again concentrated. After further dilution with ethanol, thesolutions is cooled down and the insoluble precipitates are filteredout. The resultant solution is concentrated, dried and finely ground inorder to recover the flavonoid extract in the form of a homogenouspowder.

Study of the anti-elastasic and anti-inflammatory activity of aflavonoid extract of ginkgo biloba substantially devoid of terpene

1—Study of anti-elastasic activity ex vivo

The purpose of this study is therefore to study the anti-elastasicactivity of an extract as defined above, alone or in combination withceramides. This activity is evaluated on sections of human gums.

The ceramides used, of vegetable origin, are obtained according to aprocess as described in Patent FR 2676936 incorporated by way ofreference, a process which comprises exctraction from wheat, using apolar extraction solvent followed by recrystallization from an organicsolvent. They are essentially composed of glucosylceramides andceramides.

For this purpose, the elastic fibre network is subjected to humanleucocyte elastase (HLE), with or without an anti-elastasic product. Theelastic fibres remaining after enzymatic action are stained with(+)catechin.

Frozen sections of 8 μm are carried out from 5 samples obtained frombiopsies of normal buccal mucosa, taken during dental extractions.

Protocol:

In a first stage, the concentration in human leucocyte elastase requiredfor the destruction of elastic fibres is determined: leucocyte elastaseis applied to the skin sections for two hours at ambient temperature andin a humid atmosphere; after fixing in acetone and dehydration inethanol at 70° C., the remaining elastic fibres are stained with(+)catechin. Total destruction of the elastic fibres is obtained in thepresence of 10 μm/ml of human leucocyte elastase.

In order to demonstrate any fixing of the products on the enzyme,similar quantities of products, in the form of an emulsion, and of humanleucocyte elastase at 10 μm/ml are applied simultaneously to thesections for two hours, at ambient temperature. The anti-elastasicactivity of the products is compared with that of (PMSF), a powerfulelastase inhibitor.

Morphometric quantification—by image analysis—produces the results whichare shown in Table 1 below.

TABLE 1 % of elastic fibres remaining after treatment with humanleucocyte elastase in the presence of a ginkgo extract alone or incombination with ceramides upper medium lower corium corium coriumdermis dermis dermis HLE alone 0 0 0 HLE + PMSF 25 55 70 Ginkgoextract + Ceramide (in %) (in %) 0 0.1 0 0 27.65 0 0.2 0 0 22.50 0.1 0 00 13.85 0.3 0 0 0 33 0.5 0 2.5 31.85 61.20 0.1 0.1 0 0 24.5 0.3 0.1 017.6 23 0.5 0.1 56.7 47 65 0.1 0.2 0 0 15.2 0.3 0.2 0 19 30 0.5 0.2 7053.2 80

2—Study of anti-inflammatory activity

The composition tested in this study is presented in the form of a geland comprises 0.5% of ginkgo extract as defined above and 0.1% ofvegetable ceramide as defined in the anti-elastasic activity study.

The study is carried out on 20 patients suffering from mild and moderatetartaric gingivitis. The patients apply the gel twice daily for onemonth, at the level of their gingival mucosa. Clinical assessment oftheir gingival state is carried out on days D₀ and D₂₈ using a scoringsystem. The main evaluation criteria are an assessment of erythema andgingivitis. The anti-inflammatory action of the treatment must bedemonstrated by a reduction in erythema and a reduction in gingivitis.

Clinically, erythema is demonstrated by an erythematous appearance (redcolour) of the gum and a high grade of gingivitis. The gum's return to anormal state, a reduction in the erythema, the gingivitis stage and theextent of the condition constitute clinical proof of anti-inflammatoryactivity.

Secondary criteria are an assessment of spontaneous pain and pain onchewing, and of bleeding (intensity and topography).

Operation of criteria:

The three main criteria indicating the extent of the inflammatoryactivity, “gingivitis”, “colour of gum” and “topography of condition”are assigned a coefficient of 2 with respect to the results expressed.

The “spontaneous pain”, “pain on chewing” and “papillary bleeding”(intensity and topography) are assigned a coefficient of 1 with respectto the results expressed.

For the evaluation of the gingivitis, the scores are as follows:

(0) if the grade is 0: gum normal, no inflammation, no change in colour,no bleeding;

(2) if the grade is 1: slight gingivitis, slight inflammation, slightalteration in the gingival surface, no bleeding;

(4) if the grade is 2: moderately extended gingivitis, moderateinflammation, erythema, oedema, bleeding on probing or applyingpressure;

(6) if the grade is 3: very extensive gingivitis, severe inflammation,pronounced erythema, oedema, tendency towards spontaneous bleeding,ulceration.

For the evaluation of the colour of the gum, the scores are as follows:

(0) normal

(2) slight erythema

(4) moderate erythema

(6) severe erythema

Topographical evaluation of the colour of the gum:

Six sectors are defined: RiUp, FrUp, LeUp, RiLo, FrLo and LeLo (Fr, Up,Lo, Ri and Le respectively signify Front, Upper, Lower, Right and Left).

(0) no sectors affected

(2) one sector affected

(4) two sectors affected

(6) three sectors affected

(8) more than three sectors affected

For the evaluation of papillary bleeding (Engelberger et Coll., 1993),the scores are as follows:

(0) if the grade is 0: no bleeding 10 to 30 seconds after probing

(2) if the grade is 1: slight bleeding (fine trickle of blood)

(4) if the grade is 2: moderate bleeding

(6) if the grade is 3: intense bleeding

Topographical evaluation of papillary bleeding:

(0) no sectors affected

(1) one sector affected

(2) two sectors affected

(3) three sectors affected

(4) more than three sectors affected

Changes in the “spontaneous pain and pain on chewing” criteria areevaluated using a visual scale on D₀ and D₂₈:

(0) if no pain or slight pain (measurement between 0 and 2.5 cm on thevisual scale)

(1) if moderate pain (measurement between 2.5 and 5 cm on the visualscale)

(2) if significant pain (measurement between 5 and 7.5 cm on the visualscale)

(3) if severe pain (measurement between 7.5 and 10 cm on the visualscale)

An overall score is then calculated by adding together the individualscores.

a return to a normal state corresponds to a score of 0.

maximum gravity corresponds to a score of 33.

The results are set out in Table 2 below.

TABLE 2 Day D₀ Day D₂₈ Evaluation of gingivitis 3.50 ± 1   1.0 ± 1.2 (p< 0.05) Evaluation of the colour of the gum (erythema) 4.00 ± 1.4 1.1 ±1.45 (p < 0.05) Topography of gingival 4.80 ± 1.7 1.6 ± 11.8 (p < 0.05)erythema Evaluation of papillary 0.85 ± 0.8 0.1 ± 0.43 (p < 0.05)bleeding Topography of papillary  1.20 ± 1.12 0.1 ± 0.43 (p < 0.05)bleeding Evaluation of spontaneous 1.35 ± 1.2 0 (p < 0.05) painEvaluation of pain on  1.10 ± 1.17 0 (p < 0.05) chewing Overall score16.80 ± 4.8  3.9 ± 4.6 (p < 0.05)

This study on 20 patients suffering from tartaric gingivitisdemonstrated the anti-inflammatory activity of a ginkgo extract asdefined previously in combination with vegetable ceramides.

This anti-inflammatory activity takes the significant clinical form of adisappearance of gingivitis and gingival erythema in 11 cases out of 20and an improvement in 8 other cases.

A signficant improvement in the score for papillary bleeding and acomplete disappearance, in all cases, of gingival pain (spontaneous andon chewing) were observed.

What is claimed is:
 1. A pharmaceutical composition intended for thetreatment of the dentibuccal sphere and containing as active ingredienta sufficient amount of a flavonoid extract of ginkgo biloba leavescomprising 28 to 35% by weight of flavonoid glycosides and substantiallyfree of terpenes, said extraction being several stages of extractionwith solvents with one extraction being a deterpenation extract with acompound of the formula

wherein R and R′ are independently lower alkyl optionally admixed with asaturated aliphatic hydrocarbon of at least 5 carbon atoms to treataffections thereof and an inert carrier.
 2. Composition according toclaim 1, characterized in that it exhibits an anti-elastasic activity.3. Composition according to claim 1, characterized in that it exhibitsan anti-inflammatory activity.
 4. A cosmetic composition intended forthe dentibuccal sphere and containing a flavonoid extract of ginkgobiloba leaves comprising 38 to 35% by weight of flavonoid glycosides andsubstantially free of terpenes, said extraction being several stages ofextraction with solvents with one extraction being a deterpenationextraction with a compound of the formula

wherein R and R′ are independently lower alkyl optionally admixed with asaturated aliphatic hydrocarbon of at least 5 carbon atoms to treataffections thereof.
 5. A composition of claim 1 containing 28 to 32% byweight of flavonoid glycosides.
 6. A composition of claim 1 wherein theextract contains a maximum of 1% by weight of terpenes.
 7. A compositionof claim 1 wherein the extract contains a maximum of 0.5% by weight ofterpenes.
 8. A composition of claim 1 containing 0.05 to 0.6% by weightof said extract.
 9. A composition of claim 1 also containing 0.05 to0.3% by weight of ceramides.
 10. A composition of claim 1 in the form ofa gel or a chewing gum.